Biomimetic Enzymatic Oxidative Coupling of Barley Phenolamides: Hydroxycinnamoylagmatines

Oxidative coupling of hydroxycinnamoylagmatines in barley (Hordeum vulgare) and related Hordeum species is part of the plant defense mechanism. Three linkage types have been reported for hydroxycinnamoylagmatine dimers, but knowledge on oxidative coupling reactions underlying their formation is limited. In this study, the monomers coumaroylagmatine, feruloylagmatine, and sinapoylagmatine were each incubated with horseradish peroxidase. Their coupling reactivity was in line with the order of peak potentials measured: sinapoylagmatine (245 mV) > feruloylagmatine (341 mV) > coumaroylagmatine (506 mV). Structure elucidation of fourteen in vitro coupling products by NMR and MS revealed that the three main linkage types were identical to those naturally present in Hordeum species, namely, 4-O-7′/3-8′, 2-7′/8-8′, and 8-8′/9-N-7′. Furthermore, we identified two linkage types that were not previously reported for hydroxycinnamoylagmatine dimers, namely, 8-8′ and 4-O-8′. We conclude that oxidative coupling by horseradish peroxidase can be used for biomimetic formation of natural antifungal hydroxycinnamoylagmatine dimers from barley.


HRP activity
. Absorbance of ABTS in a HRP-H2O2 system measured at 405 nm in triplicate. The enzyme activity was determined based on the average of these three measurements.   The selected fractions were most suitable for further purification, as these pools contained only two or three main compounds and contained enough material. Pools that were not selected either contained a too small amount of material or were mainly composed of the monomeric precursor. The following gradients were used for preparative chromatography of the selected pools.  Compounds (12) and (13) were not separated by preparative chromatography and were thus measured as a mixture. NMR resonances could be assigned to either one of the compounds by using HSQC and HMBC data ( Figure S8 and S9).
Both the proton and carbon NMR spectrum for feruloylagmatine differ in two positions from the spectra obtained for this compound, being two (trans)-olefinic protons at [δH 7.42 (1H, d, J = 15.7 Hz), δH 6.56 (1H, d, J = 15.7 Hz) and for carbon δC 139.0, 119.0] 1 . These signals are missing in this dimer, however one divergent proton and two divergent carbon signals are present in the spectra obtained for the dimer, being a singlet at [δH 7.90 (2H, s) linked to δC 140.78] and a resonance at δC 126.66. The loss of one of the proton signals of one of the protons linked to the carbon atoms present in the alkane group indicated that one of these carbons is involved in the linkage formed. Since no other positions showed a similar sign of substitution, it was concluded that the dimer formed is symmetrical. This indicates a symmetrical dimer was formed by an 8-8′-linkage, since HMBC showed a correlation between δC 140.78 and H-2 and H-6, proving that the proton resonance corresponds to H-7. This is comparable to the 1 H and 13 C NMR spectra for cannabisin G, an 8-8′-linked lignanamide present in Cannabis sativa 2 . Figure S8.

FerAgm-4-O-7′/3-8′-DFerAgm (18).
Compound 18  Since three signals corresponding to methoxy groups were detected it was concluded that one of the two rings was no longer symmetrical and therefore must be involved in the linkage type formed. This was also indicated by the presence of a pentasubstituted ring. In the 1 H data both (trans)-olefinic proton pairs from the sinapoylagmatine precursor were no longer detected, and from the HMBC data it could be observed that C-2 correlates with both H-7′ and H-8′. Altogether, this showed that a ring shaped linkage was formed involving C-2, C-8, C-7′, and C-8′. This is the same linkage as identified for compound 13, which is also reflected by the similarities in both the 1 H and 13 C NMR spectra. For compound 25 the signal obtained was lower, resulting in the lack of signal for C-1 and C-4′. However, based on the correlations in the HMBC data and the similarities with the data for compound 13, this linkage type could still be identified as a 2-7′/8-8′-linkage. This linkage type was also reported for a sinapoyltyramine homodimer called corydalisin C, which had 1 H and 13 C NMR spectra similar to the spectra obtained for compound 25. 5

SinAgm-8-8′-SinAgm (27).
Compound 27  , and a resonance at δC 127.43. Since one proton resonance is lost compared to the monomeric precursor and no other shifts were observed, it was concluded that this dimer is also linked by an 8-8′-linkage, resulting in a symmetrical dimer.

SinAgm-8-8′/9-N-7′-DSinAgm (29).
Compound 29 (δC 169.75, 171.80). Since two resonances corresponding to methoxy groups were observed, it was concluded that the ring of both sinapoylagmatine precursors was uninvolved in the coupling, as this would have resulted in loss of symmetry. By HMBC it could be observed that H-7 correlates with C-8′, however no correlation was found between H-7 and C-7′, indicating a linkage was formed between C-8 and C-8′. Since the typical signal for (trans)-olefinic protons was not detected by NMR, it was concluded that the double bond between C-7′ and C-8′ was saturated upon coupling, which hints at a lactam shaped linkage type, since no additional proton was observed for C-7′. In literature this linkage type was reported before for murinamide B, a feruloylagmatine homodimer. The reported 1 H and 13 C NMR spectra match with the spectra obtained for this compound, with one difference being the presence of an additional methoxy group on both benzene rings. 3 It was therefore concluded this dimer possessed an 8-8′/9-N-7′-linkage. Figure S13. 1 H (600 MHz) and 13 C (150 MHz) NMR spectra and HMBC (blue/turquoise) and HSQC (red) correlations for . Impurities in the spectrum are shown in grey (HMBC) and brown (HSQC). Table S1. UHPLC-PDA-ESI-IT-MS data and ESI-FT-MS data for the hydroxycinnamoylagmatine oxidative coupling products after 120 minutes incubation with HRP. Only fragments with a relative abundance above 10% are given. Peak numbers correspond to numbers given in Figure 2 in the manuscript.